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Impaired Macrophage and Satellite Cell InfiltrationOccurs in a Muscle-Specific Fashion Following Injury inDiabetic Skeletal MuscleMatthew P. Krause1, Dhuha Al-Sajee1, Donna M. D’Souza1, Irena A. Rebalka1, Jasmin Moradi1,
Michael C. Riddell2, Thomas J. Hawke1,2*
1Department of Pathology & Molecular Medicine, McMaster University, Hamilton, Ontario, Canada, 2Muscle Health Research Centre, York University, Toronto, Ontario,
Canada
Abstract
Background: Systemic elevations in PAI-1 suppress the fibrinolytic pathway leading to poor collagen remodelling anddelayed regeneration of tibialis anterior (TA) muscles in type-1 diabetic Akita mice. However, how impaired collagenremodelling was specifically attenuating regeneration in Akita mice remained unknown. Furthermore, given intrinsicdifferences between muscle groups, it was unclear if the reparative responses between muscle groups were different.
Principal Findings: Here we reveal that diabetic Akita muscles display differential regenerative responses with the TA andgastrocnemius muscles exhibiting reduced regenerating myofiber area compared to wild-type mice, while soleus musclesdisplayed no difference between animal groups following injury. Collagen levels in TA and gastrocnemius, but not soleus,were significantly increased post-injury versus controls. At 5 days post-injury, when degenerating/necrotic regions werepresent in both animal groups, Akita TA and gastrocnemius muscles displayed reduced macrophage and satellite cellinfiltration and poor myofiber formation. By 10 days post-injury, necrotic regions were absent in wild-type TA but persistedin Akita TA. In contrast, Akita soleus exhibited no impairment in any of these measures compared to wild-type soleus. In aneffort to define how impaired collagen turnover was attenuating regeneration in Akita TA, a PAI-1 inhibitor (PAI-039) wasorally administered to Akita mice following cardiotoxin injury. PAI-039 administration promoted macrophage and satellitecell infiltration into necrotic areas of the TA and gastrocnemius. Importantly, soleus muscles exhibit the highest inducibleexpression of MMP-9 following injury, providing a mechanism for normative collagen degradation and injury recovery inthis muscle despite systemically elevated PAI-1.
Conclusions: Our findings suggest the mechanism underlying how impaired collagen remodelling in type-1 diabetes resultsin delayed regeneration is an impairment in macrophage infiltration and satellite cell recruitment to degenerating areas; aphenomena that occurs differentially between muscle groups.
Citation: Krause MP, Al-Sajee D, D’Souza DM, Rebalka IA, Moradi J, et al. (2013) Impaired Macrophage and Satellite Cell Infiltration Occurs in a Muscle-SpecificFashion Following Injury in Diabetic Skeletal Muscle. PLoS ONE 8(8): e70971. doi:10.1371/journal.pone.0070971
Editor: Atsushi Asakura, University of Minnesota Medical School, United States of America
Received January 14, 2013; Accepted June 26, 2013; Published August 12, 2013
Copyright: � 2013 Krause et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors would like to thank: the Natural Sciences and Engineering Research Council of Canada (NSERC) for their financial support (T.J.H.), theOntario Graduate Scholarship Program (to M.P.K.) and the Canadian Institutes of Health Research Banting and Best Doctoral Fellowship Program (to D.M.D.). Thefunders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: [email protected]
Introduction
Type 1 diabetes mellitus (T1DM) is an autoimmune disease
defined by hyperglycemia and hypoinsulinemia in the absence of
exogenous insulin treatment. Unfortunately, exogenous insulin
alone does not represent a cure, but rather a therapy that fails to
completely normalize the widespread disturbances in growth and
energy metabolism. In the absence of a true cure, it is the
complications of diabetes that define the overall health of the
affected person. One such complication that has recently received
attention is diabetic myopathy [1–4]. The pathophysiology of
diabetic myopathy includes impaired muscle growth, as well as
immediate and progressive loss of muscle mass and contractile
function [1,2,4–8]. The evidence to date suggests this is, in part,
due to an imbalance between protein synthesis and proteolysis in
the diabetic muscle [9–11] and is observed as a reduction in
muscle mass and myofiber cross-sectional area. While the above
situation is concerning in terms of muscle growth, another series of
complications arise in response to muscle overuse or to muscle
injury, where a multifaceted regenerative process is needed to
repair/replace damaged myofibers. In this case, a severe
impairment in muscle repair is observed in the skeletal muscle of
T1DM; a distinct problem that is not related to any imbalances in
protein metabolism [2,12]. From a clinical perspective, as
increased physical activity is now recognized as a critical
therapeutic tool for most T1DM patients [13], defining the
capacity (and underlying mechanisms for impairments) of skeletal
muscle to grow, adapt and repair from exercise and other stressors
in diabetes becomes of paramount importance.
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The repair of skeletal muscle is a complex orchestration of
events including infiltration of immune and muscle stem cells (also
termed satellite cells), degeneration or repair of damaged
myofibers, extracellular matrix (ECM) remodelling, and formation
and subsequent growth of new myofibers [14]. The activity of
plasminogen activators (PA), such as urokinase PA (uPA), are
requisite for skeletal muscle regeneration [15–19], but PA activity
is limited by plasminogen activator inhibitor-1 (PAI-1), a diurnally
regulated hormone that is significantly and chronically elevated in
both type 1 and type 2 diabetes [2,20–22]. Recently, our lab
demonstrated PAI-1 as a mediator of the delayed muscle
regeneration in the Akita mouse model of T1DM. Pharmacolog-
ical reductions in PAI-1 activity (through PAI-039) reduced
collagen levels and restored muscle regeneration [2]. PAI-039
treatment increased active uPA and matrix metalloproteinase-9
(MMP-9) levels in Akita mouse tibialis anterior (TA) muscles,
supporting the hypothesis that PAI-1 suppresses the proteolytic
activity of uPA and MMP-9 in diabetes, thereby impairing the
regenerative process [2].
Though we, and others, have shown that PAI-1 suppresses uPA
activity, and ultimately muscle regeneration [2,19], the specific
mechanism(s) underlying how impaired collagen degradation was
delaying regeneration remained unanswered. We hypothesized
that excessive collagen levels in the muscle of Akita mice would
attenuate infiltration of macrophages and satellite cells into the
damaged areas leading to a delay in muscle regeneration.
Furthermore, in rodent studies of T1DM, muscle groups with a
high proportion of glycolytic myofibers [such as the TA,
gastrocnemius, and extensor digitorum longus] appear to suffer
the greatest myofiber atrophy, while the soleus consistently
demonstrates resilience to diabetic myopathy [4,6–8,23–26].
Thus, we further hypothesized that there would be muscle
group-specific differences observed in terms of inflammatory and
satellite cell infiltration and regenerative capacity with the
resilience of the soleus extending to the process of muscle repair.
Our findings support the hypothesis that PAI-1 mediated delay in
ECM remodelling leads to a delay in macrophage and satellite cell
infiltration into the damaged areas of diabetic skeletal muscle. We
also demonstrate that although the increase in PAI-1 is systemic,
intrinsically higher inducible expression of MMP-9 in response to
injury within the oxidative, postural muscles (soleus) versus more
glycolytic, non-postural muscles (TA) provides a mechanism for
the soleus (and other oxidative) muscles to maintain normal ECM
turnover during injury and ultimately maintain normal regener-
ative capacity.
Methods
Animal CareAll animal experiments were approved by the McMaster and
York University Animal Care Committees in accordance with
Canadian Council for Animal Care guidelines.
Male C57BL/6-Ins2Akita/J (hereafter referred to as Akita) mice
and their wild-type littermates (WT) were purchased at 3 weeks of
age from Jackson Laboratory (Bar Harbor, ME). Akita mice and
WT mice (n = 12) were studied over a period of up to 8 to 13
weeks of untreated T1DM (depending on time-point of regener-
ation measured). Akita mice become spontaneously diabetic at ,4
weeks of age due to a heterozygous mutation in the Ins-2 gene.
Monitoring of diabetes onset was performed as described
previously [2,4].
To determine if restoration of PAI-1 activity in the diabetic
mouse would normalize macrophage infiltration into the regen-
erating TA, an additional group of WT and Akita mice (n = 4)
were treated via oral gavage with vehicle (2% Tween-80 and 0.5%
methylcellulose in sterile H2O) or vehicle plus PAI-039 (2 mg/kg;
Axon Medchem, Netherlands, axon1383), an orally effective
inhibitor of active PAI-1 [27,28]. On the day of CTX injury (at 8
weeks of diabetes, or approximately 12 weeks of age), the mice
were treated with vehicle or vehicle plus PAI-039 at 1100 hours,
then received CTX injury to the TAs with 10 uM CTX at 1200
hours, and received PAI-039 treatment again at 1500 hours. PAI-
039 treatment was continued twice daily at those designated times
throughout the 5 day regeneration period, at which point the
animals were sacrificed and tissues were dissected and stored as
described below. Those treatment time-points were chosen to best
attenuate the peak of PAI-1 activity due to its circadian expression
pattern. As well, as collagen levels within the resting skeletal
muscle of these young adult Akita and WT mice were not different
(not shown), administration of PAI-039 began 1 hour before injury
as there was not a need to act on the established extracellular
matrix. WT mice treated with vehicle (WT+vehicle) demonstrated
no significant difference from untreated WT in any variable
measured and these groups were pooled for comparison with Akita
mice and Akita mice treated with PAI-039 (Akita+PAI-039).Blood glucose and body mass were measured biweekly with
animals in the fed state. Blood samples were collected at 6 weeks of
Table 1. Characteristics of wild type and Akita diabetic mice.
Measure Group Time of diabetes
6 weeks 9–13 weeks
Body Mass (g) WT 24.9 (0.4) 27.7 (0.1)
Akita 22.9 (0.4)* 24.5 (0.1)*
Insulin (pg/ml) WT 814 (106) 1650 (218)
Akita 222 (56)* 209 (24)*
Blood Glucose (mM) WT 9.1 (0.3) 8.7 (0.2)
Akita 32.4 (0.7)* 32.2 (0.4)*
NEFA (mM) WT 1.10 (0.06) 0.66 (0.04)
Akita 2.14 (0.12)* 1.59 (0.12)*
PAI-1 (pg/ml) WT 429.6 (112.0) 291 (33)
Akita 1593.5 (134.3)* 960 (217)*
TA Mass (mg) WT 50 (2)
Akita 41 (2)*
GPS Mass (mg) WT 177 (6)
Akita 141 (3)*
TA fiber area (mm2) WT 2285 (92)
Akita 1851 (36)*
GAS fiber area (mm2) WT 2314 (102)
Akita 1926 (83)*
SOL fiber area (mm2) WT 1154 (47)
Akita 1094 (41)
Data collected at 6 weeks of diabetes (6 weeks group), or at 5, 10, or 35 daysfollowing cardiotoxin muscle injury at 8 weeks of diabetes (9–13 weeks group).All measures except for soleus fiber area were found to be significantly alteredin the diabetic mice. All muscle mass and fiber area measures presented hereare from the uninjured muscle; the cardiotoxin injured contralateral leg muscledata are presented in Figure 1. NEFA, non-esterified fatty acids; PAI-1,plasminogen activator inhibitor-1; TA, tibialis anterior; GPS, gastrocnemius-plantaris-soleus complex; GAS, gastrocnemius; SOL, soleus. * denotes significantdifference in Akita compared to matching wild type (WT) value as assessed by t-test (P,0.05).doi:10.1371/journal.pone.0070971.t001
Muscle-Specific Effects in Type 1 Diabetes
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diabetes via tail-nick for analysis of metabolites, hormones and
other cytokines. The animal room was maintained at 21uC, 50%humidity and 12 h/12 h light-dark cycle. All mice had access to
standard breeder chow, water ad libitum and enrichment material.
Figure 1. Soleus muscle is resilient to the regenerative defects found in other muscles from Akita mice. (A) H&E stained cryosections oftibialis anterior (TA), gastrocnemius (GAS), and soleus (SOL) illustrate decrements in muscle regeneration in diabetic Akita mouse muscles. (B) TAdemonstrates the worst impairment in regeneration, as determined by cross sectional area of regenerating (centrally-nucleated) fibers (significantmain effect of diabetes and interaction [P,0.05]). GAS (C) also demonstrates impaired regeneration (significant main effect of diabetes andinteraction [P,0.05]), while SOL (D) does not. In panels E–F, TA (E), GAS (F), and SOL (G) CTX-injured fiber area data are expressed relative to fiber areafrom the contralateral uninjured muscles. The TA (significant main effect of diabetes and interaction [P,0.05]) and GAS (significant main effect ofdiabetes [P,0.05]) both demonstrate poor regeneration even when expressed in these relative terms. The mass of muscles that had been injuredwith cardiotoxin further illustrate poor regeneration. The muscle mass of the injured TA (H; significant main effect of diabetes and interaction[P,0.05]) and gastrocnemius-plantaris-soleus (GPS) complex (I; significant main effect of diabetes [P,0.05]) are illustrated here. * denotes significantpost-hoc analysis differences (P,0.05). Scale bar represents 50 um.doi:10.1371/journal.pone.0070971.g001
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Skeletal Muscle Injury and Tissue CollectionSkeletal muscle injury was induced following 8 weeks of diabetes
(approximately 12–13 weeks old) via an intramuscular injection of
100 mL of 10 mM cardiotoxin (CTX; Latoxan, Valence, France)
per muscle, as previously described [29]. Injuries were generated
in the left TA and gastrocnemius-plantaris-soleus (GPS) muscles of
both Akita and WT mice. These mice were divided into 3 groups
to allow different lengths of recovery (5, 10, or 35 days post injury).
Following the specified regeneration period, animals were
euthanized by CO2 inhalation and cervical dislocation, and tissues
were collected and stored appropriately for future analyses. When
muscles were dissected, both CTX-treated and untreated TA and
GPS muscle groups were weighed for comparison.
Blood AnalysesHeparinized blood plasma was analyzed for insulin and total
PAI-1 (Multiplex Adipokine assay from Millipore, MADPK-71K)
at all collection time points. Plasma was also analyzed for non-
esterified fatty acids (NEFA) with the use of a colorimetric assay
(NEFA-HR2, Wako diagnostics, Wako TX).
Histochemical and Immunofluorescent AnalysesFrozen OCT-embedded skeletal muscle was cut using a
cryotome into 8 mm cross-sections and mounted on untreated
glass slides for histochemical or immunofluorescent staining.
H&E staining. Hematoxylin and eosin staining was used to
determine average cross-sectional area of uninjured and injured
myofibers. Three images spaced evenly throughout the TA were
used for analysis where 25 fibers per image were analysed for
mean fiber area (75 total fibers per TA). In the soleus and
gastrocnemius, between 50–75 fibers were analysed to represent
fiber area, as has previously been validated [30,31].
Picrosirius red staining. To stain for collagen content,
picrosirius red staining was performed as previously described
[2,32]. The myofibers appear yellow while the collagen retains the
red stain, providing adequate contrast for collagen [2].
Immunofluorescent Staining. Muscle section preparation
was performed as previously described [2]. To identify Myh3-
expressing myofibers, sections were incubated with 1:1 dilution of
anti-Myh3 (F1.652; DSHB, Iowa City, IO) in blocking buffer
1.5% normal goat serum and 1.5% horse serum albumin)
overnight at 4uC. Similarly, F4/80 antigen was used to identify
macrophages in injured muscles, using a dilution of 1:200 F4/80
antibody (AbD Serotec, CedarLane Labs, Burlington, ON),
however, mouse IgG block was omitted. Appropriate secondary
antibody application was performed (Alexa 488 anti-mouse
antibody [Invitrogen, A-11001] or Alexa 594 anti-rat antibody
[Invitrogen, A-11007]) followed by 5 min of 1:1000 4,6-diami-
dino-2-phenylindole (DAPI) to identify nuclei. To facilitate
identification of positively stained myofibers or macrophages,
either laminin or type I collagen was stained for using anti-laminin
(ab14055, Abcam, Cambridge, MA) or anti-collagen, type I
(ab292, Abcam). Satellite cells were identified by immunostaining
for Pax7. Freshly cut 5 mm sections were air-dried, then fixed for 4
minutes in ice-cold 2% PFA. Sections were then incubated in 1%
triton X-100 for 30 minutes followed by blocking buffer for 30
minutes. Pax7 antibody (DSHB) was diluted 1:1 in a blocking
buffer and incubated overnight at 4uC. A biotin-streptavidin
(Vector Labs, Burlington, ON) detection system was used with
Dylight 594 (Abcam, 1:250 in blocking buffer) used to visualize
Pax7 positive nuclei with a DAPI co-stain.
Image AnalysisImages of stained sections were obtained with a Nikon 90i
eclipse upright microscope (Melville, NY) and analyzed using
Nikon Elements software. Analysis included determination of
collagen, Myh3, and F4/80 positive area using signal threshold
settings as the detection method. In all staining procedures, a
negative control (no primary antibody) was performed and used to
set the signal threshold settings. In all cases, negative controls
displayed an absence of signal. For determination of F4/80 cell
number, the threshold binary for DAPI-stained nuclei was
intersected with the F4/80 threshold binary; the number of
intersected objects represented the positive cell number. Regen-
erating (injured) and uninjured muscle fiber area in H&E stains
was determined manually using Elements software. Both regen-
Figure 2. Tibialis anterior muscle of Akita mice demonstrate transient, excessive fibrosis during early regeneration. (A) TAdemonstrates elevated collagen during regeneration, particularly at 5 days post-injury (significant main effect of diabetes [P,0.05]), whilegastrocnemius (GAS) (B) demonstrates a non-significant trend (main effect of diabetes [P = 0.11]). Conversely, soleus (SOL) (C) does not demonstratedysregulated collagen expression. * denotes significant post-hoc analysis differences (p,0.05). (D) Representative picrosirius red staining of 5 and 10day post-injury TA muscles. Note the elevated presence of collagen (red) and smaller size of myofibers (yellow) in Akita TA. Scale bar represents50 um.doi:10.1371/journal.pone.0070971.g002
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erating fibers, easily differentiated from uninjured fibers due to
their small size with centrally-located nuclei, and uninjured fibers
were manually encircled using the software thus identifying their
cross-sectional area. TA, gastrocnemius, and soleus muscles were
analyzed separately to determine muscle group-specific differenc-
es. Regenerating and necrotic injured muscle tissue represent
different stages of the overall regeneration process, and, thus, were
identified in immunofluorescently stained tissue and analyzed
separately as specifically illustrated within our figures and similar
to the analyses conducted previously [33].
ImmunoblottingHomogenates of soleus, gastrocnemius and TA muscles from
WT mice were prepared as before [2]. Fifteen micrograms of
protein sample were loaded, resolved by 10% SDS-page gels, and
transferred to PVDF membranes. Primary antibodies for total
MMP-9, (1:5000, Abcam) and beta-actin (1:2500, Abcam) were
used to detect proteins of interest, and were subsequently
conjugated with the appropriate horseradish peroxidase secondary
antibodies. Signals were visualized using chemiluminescent
reagent (GE Healthcare, Baie d’Urfe QC), and acquired with
the CareStream imager and accompanying software (Woodbridge,
CT).
Statistical AnalysisFor all experiments, the appropriate t-test or two-way ANOVA
with Bonferroni post-hoc analysis of pairwise comparisons was
carried out to classify significant differences (P,0.05) between
Akita and WT groups. Two-way ANOVA was run on data sets
with dependent variables measured over time, while one-tailed t-
tests were carried out on data with only single comparisons. Data
are presented as mean6SEM. An * denotes a significant difference
identified by t-test or Bonferonni post-hoc test in pairwise
comparisons, while a significant main effect of diabetes or a
significant interaction between diabetes and time are listed within
figure legends.
Results
Akita mice spontaneously developed hypoinsulinemia and
hyperglycemia at ,4 weeks of age, which was maintained
throughout the experiments (Table 1), consistent with previous
findings [2,4,34]. Akita mice also demonstrated hyperlipidemia,
elevated PAI-1 and attenuated body mass accrual by 6 weeks of
untreated diabetes (Table 1), consistent with observations of
T1DM in humans [20,34,35] and diabetic rodents
[2,21,22,31,36,37].
Figure 3. Necrosis of muscle fibers persists throughout muscle regeneration in Akita tibialis anterior muscle. (A) Uninjured (left),necrotic (center), and actively regenerating (right) regions of skeletal muscle are easily identified in collagen type I immunostain (green) with DAPI(blue) counterstain. Note the presence of centrally located nuclei in muscle fibers in regenerating muscle, indicative of new myofiber formation. (B)TA of diabetic mice have clearly defined areas of necrosis remaining at 10 days post-injury (significant interaction [P,0.05]; * denotes post-hocdifference). Similarly, gastrocnemius (GAS) (C) follows this pattern although not statistically significant (P = 0.21). In contrast, both WT and Akita soleus(SOL) display no areas of necrosis at 5 or 10 days post-injury (not shown). (D) Representative image of TA muscle undergoing regeneration at 10 dayspost-injury in WT and Akita TA. Note the distinct area of necrosis in the Akita TA. Scale bar represents 500 um.doi:10.1371/journal.pone.0070971.g003
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Assessment of the uninjured leg of Akita mice revealed
significantly less muscle mass in the TA and GPS compared to
WT (Table 1). Furthermore, mean myofiber area was reduced in
Akita mice compared to WT in H&E stained cross-sections of TA
and gastrocnemius. However, uninjured soleus of Akita mice
exhibited no difference with WT in myofiber area (Table 1),
consistent with previous studies [6–8,23,24]. Though soleus
muscles were not weighed separately, given no difference in fiber
area, we would hypothesize no difference between groups in soleus
mass.
In response to cardiotoxin injury, Akita TA and gastrocnemius
exhibited a significant loss of myofiber area in regenerating
muscles compared to WT (Figure 1A–C), even when expressed
relative to uninjured fiber area from the contralateral TA and
gastrocnemius, respectively (Figure 1E, F). Conversely, soleus did
not demonstrate a reduction in regenerating fiber area, in absolute
or relative terms (Figure 1D, G). Masses of the injured TA and
GPS complex were reduced in Akita mice compared to WT as
well (Figure 1H, I), corresponding with the reductions in myofiber
areas observed in the gastrocnemius and TA muscles.
Given this muscle-specific diversity in responses to the diabetic
environment, further investigation into the regeneration process of
Akita diabetic muscle was needed. It is known that ECM
remodelling is a critical component of the regeneration process
[2,19,31,38], thus, we determined the expression levels of a
primary ECM component, collagen. The injured TA muscles of
Akita mice were found to have elevated collagen expression
compared to WT, particularly at 5 days post-injury, as demon-
strated by picrosirius red staining (Figure 2A, D). While the
regenerating gastrocnemius exhibits a non-significant trend
(P= 0.11), the regenerating soleus (P = 0.51) muscle does not
demonstrate significant differences in collagen expression between
Akita and WT mice (Figure 2B–C). The picrosirius red stained TA
muscles also clearly demonstrate smaller fiber areas (yellow
staining) observable at 10 days post-injury (Figure 2D), consistent
with the analysis of H&E stained tissue sections.
Though the increased expression of collagen during the early
phase of regeneration is important to maintain muscle integrity as
the myofibers regenerate, we speculated that excessive collagen
levels, particularly in the Akita TA muscles would attenuate the
normal regenerative process. More specifically, we hypothesized
that excessive collagen levels would delay the degeneration phase;
a hypothesis that was tested by evaluating the size of necrotic
versus regenerating areas within regenerating Akita and WT
muscles. Sections of TA and GPS were immunofluorescently
stained for type I collagen and DAPI so as to accurately identify
necrotic and regenerating areas in each muscle (Figure 3A). Areas
with highly disrupted collagen surrounding large, rounded
(hypercontracted) myofibers were identified as damaged muscle
undergoing necrosis, while areas with small, centrally-nucleated
Figure 4. Macrophage and satellite cell infiltration into necrotic muscle is impaired in Akita diabetic mice at 5 days post injury. (A)The necrotic area of the TA demonstrates a reduced number of macrophages as evidenced by less F4/80 positive cells. * denotes significantdifference by t-test (p,0.05). (B) A similar, though non-significant, trend of attenuated F4/80 positive cells was observed in necrotic gastrocnemius(GAS) (P = 0.09). (C) Representative images of necrotic regions of TA and GAS immunofluorescently stained for F4/80 (red), costained for type Icollagen (green) and nuclei with DAPI (blue). Satellite cells (Pax7+ cells) were also reduced in number within necrotic areas of (D) Akita TA and (E) GAS(P = 0.06). (F) Representative images of Pax7 positive cells within necrotic regions of WT and Akita TA. Scale bar represents 50 um.doi:10.1371/journal.pone.0070971.g004
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Figure 5. Initiation of muscle regeneration is delayed in tibialis anterior and gastrocnemius muscles in Akita mice. Regions of necroticmuscle tissue in (A) TA and (B) gastrocnemius (GAS) demonstrate attenuated response of Myh3-expressing myofibers in diabetic mice. An * denotes a
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myofibers represented areas of regeneration. In each muscle, the
sum area of the necrotic regions was determined and expressed as
a percentage of the total injured area (necrotic area+regeneratingarea) in the muscle. The soleus did not have any remaining
necrotic regions at 5 days post-injury in WT or Akita mice,
whereas the TA and gastrocnemius exhibited varying amounts of
necrosis at 10 days post-injury (Figure 3B–D). The TA of WT
mice demonstrated a larger necrotic region size at 5 days post-
injury (P,0.05) compared to the Akita TA. Necrosis was absent in
the TA muscle of WT mice at 10 days post-injury, but still present
in Akita mice (significant interaction: P,0.05; Figure 3B). Though
a temporal pattern similar to that observed in the Akita TA was
observed in the gastrocnemius, it did not reach statistical
significance (P= 0.21; Figure 3C). Quantification of necrotic
region size in tissue sections (depicted in Figure 3D), was
confirmed in other sections stained for laminin and DAPI, as well
as by H&E staining (not shown).
To determine if the impaired regenerative response observed in
Akita TA and gastrocnemius muscles was a result of impaired
macrophage infiltration secondary to elevated collagen levels, we
undertook F4/80 immunostaining and quantification within the
necrotic and regenerating regions of the injured muscles. In the
Akita TA, a significant reduction in F4/80-positive cells was found
in the necrotic region at 5 days post-injury (P,0.05; Figure 4A). A
similar, but non-significant, trend (P = 0.09) was observed in the
necrotic region of the gastrocnemius (Figure 4B). In necrotic areas,
macrophages are typically found around the edge of the
degenerating myofibers or entering the degenerating fibers as
they are degraded and phagocytosed (Figure 4C). In regenerating
regions of the injured muscles (TA, gastrocnemius, soleus) no
significant difference between groups was noted in macrophage
number at 5 or 10 days post-injury (Figure S1). Importantly, no
difference in macrophage density was detected between groups in
uninjured TA (WT: 643625, Akita: 620669 cells/mm2, P= 0.43),
gastrocnemius (WT: 714646, Akita: 659664 cells/mm2, P= 0.26)
or soleus (WT: 683684, Akita: 810638, P = 0.12) indicating that
the observed impairment was in the regeneration process and not
an extension of an inherent defect in diabetic mouse skeletal
muscle or the basal, low-level, inflammatory state which charac-
terizes diabetes.
Given the delay in macrophage infiltration into the necrotic
regions of the TA and, to a lesser extent, the gastrocnemius of
Akita mice, we speculated that satellite cell infiltration into the
necrotic areas of damaged muscle may also be affected. For this
analysis, we quantified the SC number per mm2 in the necrotic,
regenerating and undamaged areas of all 3 muscle groups in the
Akita and WT mice. Firstly, no significant defect in SC number
was noted between Akita and WT mice, regardless of muscle, in
the undamaged areas of the muscles (Table S1). Consistent with
the deficit in macrophage infiltration, a significant decrease in
satellite cell number within necrotic areas of TA was noted for
Akita mice compared to control (Figure 4D), while the gastroc-
nemius exhibited a non-significant trend (P= 0.06). This impair-
ment was not observed within the Akita soleus muscles due to the
absence of necrosis at this time point. Also consistent with our F4/
80 observations, no differences between Akita and WT was noted
in SC density within regenerating areas of all 3 muscle groups
(Table S1).
An important stage of skeletal muscle regeneration involves the
de novo formation of myofibers from the fusion of myoblasts.
These nascent myofibers will initially express the embryonic
isoform of myosin heavy chain (Myh3) before expressing mature
myosin isoforms. In the necrotic region of the TA at 5 days post
injury, significantly less Myh3 expression was observed in Akita
mice compared to WT (Figure 5A, C), as well as in the
gastrocnemius (Figure 5B, C); observations that are consistent
with the reduced infiltration of muscle satellite cells. In regener-
ating regions, expression of Myh3 observed at 5 days post-injury
indicates a reduction in Akita mice (significant interaction:
P,0.05). However, at 10 days post-injury, there is still significant
expression of Myh3 in the Akita TA and gastrocnemius
(Figure 5D–E) compared to WT (P,0.05), indicating attenuated
repair as the regenerating WT muscles had already progressed to
more mature myosin isoforms [39]. No delay in Myh3 expression
was noted in the Akita soleus, consistent with no difference from
WT in collagen levels or macrophage infiltration (Figure 5F). Size
of Myh3-positive myofibers was also determined in the regener-
ating region of the three muscle groups and a similar pattern was
observed. The gastrocnemius demonstrated a delay in the growth
of the Myh3-positive cells (interaction: P,0.05), while the TA
exhibited a trend for a delay (P= 0.078; Figure S2A–B). In
contrast, the soleus did not demonstrate any delay in the growth of
Myh3-positive myofibers (Figure S2C). These data again demon-
strate a deficit in the regenerative capacity of Akita TA and
gastrocnemius, but not soleus muscles.
While a PAI-1 mediated delay in collagen breakdown is a
primary contributor to the attenuated muscle regeneration in TA
muscles of Akita mice [2], we speculated that resilience of the
soleus to the impaired regeneration in T1DM was the result of this
muscle’s intrinsically greater inducible expression of MMP-9 [40].
Consistent with previous reports [40], minimal amounts of MMP-
9 protein were observed in any of the uninjured skeletal muscles,
and no difference was observed between Akita and WT. However,
MMP-9 protein levels increased significantly post-injury, in a
muscle-specific pattern, in both Akita and WT (main effect of
muscle group: P,0.05; Figure 5H). It is also noteworthy that while
soleus muscles exhibit the greatest inducible MMP-9 expression, a
greater post-injury MMP-9 expression was observed in gastroc-
nemius compared to TA; a finding that might help explain the
intermediate phenotype this muscle shows with respect to muscle
regeneration in T1DM.
To support our hypothesis that the elevation in PAI-1 is the
upstream mediator of the delayed infiltration of macrophages and
satellite cells into the non-postural, more glycolytic muscles (TA,
significant difference detected by t-test (p,0.05). (C) Representative image of Myh3 immunofluorescent stained (red) TA and GAS necrotic regions.Co-staining of laminin (green) and nuclei with DAPI (blue) was performed. In the actively regenerating regions of the (D) TA and (E) GAS, theattenuation of Myh3 expression was not as severe, and at 10 days post-injury, was found to be significantly elevated in diabetic mice compared towild type, indicative of delayed regeneration (significant interaction [P,0.05]). The * indicates significant differences detected by post-hoc testing(P,0.05). Conversely, (F) Akita soleus (SOL) demonstrates no impairment in establishing Myh3 expression following injury and does not continue tosignificantly overexpress Myh3 at 10 days post-injury. (G) Representative image of regenerating regions of muscles at 5 days post-injury stained forMyh3 (red) and laminin (green). Inset (derived from TA muscles) is provided without green channel to clearly demonstrate Myh3 stain morphology.Scale bar represents 50 um in panels C and G. (H) MMP-9 immunobloting on uninjured and regenerating (2 days post-injury) TA, GAS and SOLmuscles from WT and Akita mice demonstrate the intrinisic differences between muscles in pro-MMP-9 expression (main effect of muscle group;P,0.05), but no differences between WT and Akita mice. MMP-9 content is relative to beta-actin loading controls and representative MMP-9 blots areincluded.doi:10.1371/journal.pone.0070971.g005
Muscle-Specific Effects in Type 1 Diabetes
PLOS ONE | www.plosone.org 8 August 2013 | Volume 8 | Issue 8 | e70971
gastrocnemius) of Akita mice, we treated diabetic Akita mice with
a PAI-1 inhibitor, PAI-039, during the regenerative process. PAI-
039 administration returned F4/80 positive cell density in the
necrotic areas of the TA muscles of Akita mice to that observed in
WT mice, though this was not statistically significant (P = 0.06;
Figure 6A, B). A similar trend was observed in the gastrocnemius:
F4/80 cell density was improved but not significantly (P = 0.07;
Figure 6C). Contrary to the deficit in Pax7 positive cells within the
necrotic areas of TA muscle at 5 days post-injury (Figure 4D), PAI-
039 treatment restored Pax7 positive cell number in the necrotic
regions of the TA and gastrocnemius (P,0.05; Figure 6D, E).
Discussion
Poorly controlled T1DM is associated with a number of
negative effects on skeletal muscle including attenuated growth
[2,3,4] and impaired regeneration [1–4,12,41]. We have demon-
strated that the TA muscles of Akita mice exhibit impaired
regeneration with as little as 7 days of diabetes exposure prior to
injury [2]. Further, we defined the primary mechanism for this
impairment as a chronic elevation of PAI-1 slowing ECM
breakdown through reductions in uPA and active MMP-9
enzymatic activity. What remained unanswered was: (1) by what
specific mechanism the attenuation of ECM turnover was delaying
regeneration and (2) whether this phenomenon was occurring
similarly in all muscle groups. This second point was most
intriguing since it has been reported that the soleus muscle of
streptozotocin-induced diabetic rodents is resistant to the negative
effects of diabetes on growth and function [6–8,23,24] but it was
not known if this extended to post-injury regeneration.
The findings of the present study reveal that exposure to the
T1DM environment results in impaired skeletal muscle regener-
ation; a finding that is more pronounced in non-postural muscles
with a greater glycolytic fiber-type composition. In the Akita TA,
and to a lesser extent the gastrocnemius, muscle injury is followed
by an excessive collagen accumulation (relative to WT). The
present results suggest that this attenuation in collagen breakdown
impairs the infiltration of macrophages and muscle satellite cells
into the areas of damage and necrosis, ultimately prolonging the
necrotic phase of muscle regeneration. Koh and colleagues [19]
previously proposed that efficient removal of the ECM is necessary
for effective macrophage and satellite cell migration during muscle
regeneration and the current findings are in line with this
hypothesis. Ultimately, this delay in degeneration translates into
an attenuation of the regenerative phase, as illustrated by a
temporal delay in the expression of developmental myosin heavy
chain isoforms and reduced myofiber sizes. Interestingly, the
soleus muscles of Akita mice did not display any of the
impairments noted above, a finding of critical importance as we
begin to unravel mechanisms underlying T1DM-mediated com-
plications within skeletal muscle.
PAI-1 has been characterized as a primary suppressor of
proteolytic activity in the extracellular space, slowing muscle
regeneration down by inhibiting a cascade of protease activation.
PAI-1 directly binds and inhibits uPA, the key plasminogen
activator in muscle regeneration, preventing active plasmin from
disrupting ECM proteins and from cleaving pro-MMP-2 and pro-
MMP-9 into their active forms. These MMPs, particularly MMP-
9, are crucial during the early stages of regeneration, functioning
to enzymatically cleave protein chains composing the ECM;
allowing for infiltration of both macrophages and satellite cells to
regions of skeletal muscle damage [15–19,42]. Indeed, the
importance of MMP-9 to skeletal muscle ECM remodelling is
demonstrated by our observations that Akita mice display reduced
active MMP-9 expression [2] and that total MMP-9 inducible
expression is greatest in soleus muscles compared to TA and
gastrocnemius at 2 days following muscle damage (Figure 5). Such
high levels are thought to promote rapid repair of the soleus,
eliminating the necrotic area and excessive collagen expression
that had been found in the glycolytic TA and gastrocnemius at 5
and 10 days following injury. It was hypothesized that intrinsically
higher, inducible, MMP-9 expression would afford the soleus of
Akita mice a mechanism by which this muscle is able to maintain
normative regeneration rates. This prediction is based on reports
of PAI-1 mediated fibrosis in cardiac and skeletal muscle [43,44]
and of a PAI-1 mediated-suppression of ECM remodelling (via
uPA and MMP-9) as the primary contributor to delayed
regeneration in the Akita TA [2].
While PAI-1 has been reported to impair macrophage
infiltration via uPA inhibition within injured muscle [15,17–
19,42], the fact that this is occurring in injured skeletal muscle of
diabetic mice is a critical finding to unlocking the mechanisms
underlying diabetic myopathy development. Macrophages play
multiple roles in the regeneration process. Initially, these cells
phagocytose damaged/dying muscle fibers and debris, but by 3–5
days post-injury, macrophages switch to a role of signalling for
myoblast fusion and differentiation [45]. The present findings
indicate that reduced macrophage infiltration in the diabetic
mouse results in necrosis persistent at 10 days post-injury
concurrent with delayed initiation of the regeneration phase
indicated by decreased satellite cell infiltration and reduced Myh3
expression. Quite simply, these deficits in the degenerative and
early regenerative phases could lead to attenuated muscle repair.
These findings are consistent with the observations of others who
have proposed that excessive collagen accumulation or impaired
collagen turnover would attenuate macrophage and possibly
satellite cell infiltration into the necrotic areas of damaged skeletal
muscle [19]. Furthermore, our results demonstrating that systemic
PAI-1 inhibition can restore the number of macrophages
infiltrating the necrotic regions of injured TA and gastrocnemius
muscles of diabetic mice to WT levels and ultimately restore the
regenerative capacity lends further support to this hypothesis.
While we believe this cascade of events accurately depicts the
deficits in Akita mouse TA muscle, differential effects were noted
between muscles groups in these diabetic mice. Of the muscle
groups studied in the current work, the TA demonstrated the most
severe impairment in regeneration, while the soleus was clearly
resistant to the diabetic environment with respect to both growth
and regeneration. The gastrocnemius exhibited an intermediate
phenotype, with impairments less severe than the TA, though
growth and regeneration were still significantly delayed. This
intermediate phenotype of the gastrocnemius is consistent with the
mixed fiber-type distribution of this muscle. The evidence to date,
derived from STZ-induced diabetic rodents, supports our findings
that the soleus exhibits resilience against diabetic myopathy.
Previous studies demonstrate that the soleus, which primarily
expresses type I myosin heavy chain [46], is resistant to the
atrophy or lack of growth induced by T1DM [6–8]. In fact,
isolated type I fibers from diabetic rats demonstrate no loss of fiber
size or contractile force [26], contrary to that found in muscles
composed primarily of type II fibers [4,7,8,23,24]. Given previous
[2] and present results, chronic elevations in PAI-1 appear to be
the upstream mediator of the impaired regeneration observed in
T1DM mice. Here we provide a mechanism as to how this rise in
systemic PAI-1 could elicit such dramatic impairments within the
TA but have little impact on the regenerative capacity of the
soleus. The answer appears to lie with the intrinsic differences
between these muscle groups in the fibrinolytic pathway. Indeed,
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Muscle-Specific Effects in Type 1 Diabetes
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during adolescent growth or following muscle injury, a greater
inducible-expression and activity of MMP-9 is observed in
oxidative muscle, such as the soleus, compared to the glycolytic
muscle [40,47]. As discussed, MMP-9 activity is critical for muscle
regeneration to occur [38,48,49], and a greater inducible-
expression in the diabetic soleus would allow repair to proceed
normally, compared to the glycolytic TA and gastrocnemius,
which lack the same degree of inducible MMP-9 expression.
Evidence also exists that collagen types I, III and IV, the main
constituents of skeletal muscle collagen, undergo a much milder
gene expression response in the injured soleus compared to injured
glycolytic muscles [50]. This implies that the soleus has a less
extensive requirement for ECM remodelling compared to
glycolytic muscle groups. Furthermore, uPA is highly expressed
in soleus but not glycolytic muscle groups [51], meaning that
despite increases in the PAI-1:uPA ratio, free (active) uPA may still
be available in the diabetic soleus, affording it the opportunity to
activate the proteolytic cascade. Thus, intrinsic differences
between muscle groups, in terms of ability to remodel the ECM
and the amount of remodelling needed, would account for the
observed differential response to injury noted in diabetic muscle
groups.
Though we have proposed a PAI-1 mediated impairment in
collagen remodelling as the primary mechanism of attenuated
regeneration in diabetic mice, an alternative scenario (which need
not be mutually exclusive) could involve impairments in macro-
phage and satellite cell proliferative/migratory capacity in Akita
mice. While this hypothesis was untested in the current study, our
present results suggest that a global defect in satellite cells or
macrophages is not likely the primary mediator of the impaired
muscle regeneration as deficits in muscle repair were not exhibited
in the soleus muscles of diabetic Akita mice. What’s more, no
difference in the number of Pax7-positive cells was noted between
groups in the regenerating soleus 5 days post injury. Finally, the
ability of PAI-039 to restore normative regeneration (as assessed
by collagen levels and Myh3 levels) [2] suggests that the
plasminogen to MMP-9 pathway is the primary defective pathway
in this diabetic model. Clearly, future studies are needed to further
investigate the effects of the T1DM environment on satellite cell
and macrophage functional capacities.
ConclusionsIn conclusion, our data support the novel hypothesis that
skeletal muscle regeneration in T1DM is characterized by poor
macrophage infiltration into damaged muscle tissue. This delay in
the degenerative phase further results in a slowed migration of
satellite cells into the necrotic areas and ultimately reducing
muscle fiber size in the regenerating muscle of type 1 diabetics.
Importantly, our findings demonstrate, for the first time, that
muscle group-specific differences exist. Intrinsic differences within
muscle groups in the proteolytic cascade (e.g. MMP-9) allow
oxidative, postural muscles, such as the soleus, to effectively
regenerate despite the negative influence of a systemic rise in PAI-
1. Future experiments unravelling the mechanisms affording the
oxidative soleus muscle resistance to other aspects of diabetic
myopathy (e.g. altered metabolism, impaired growth) will be
critical to developing therapeutic strategies aimed at maintaining
skeletal muscle health in the face of T1DM.
Supporting Information
Figure S1 Macrophage content in regenerating regionsof Akita diabetic muscles is not different from wild-typemuscles. In actively regenerating areas of the 5 day post CTX-
injured (A) TA, (B) gastrocnemius (GAS) and (C) soleus (SOL)
muscles, no significant alteration in macrophage number was
found. (D) Representative images of regenerating muscles stained
for F4/80 (red) and type I collagen (green). Inset of TA muscles is
provided without green channel to clearly demonstrate F4/80
stain morphology. Scale bar represents 50 um.
(TIF)
Figure S2 Growth of Myh3-positive cells is delayed inregenerating Akita tibialis anterior and gastrocnemiusbut not soleus muscles. The average size of Myh3-positive
objects was determined and it was found that the TA (A) exhibited
a strong trend for a delay in the growth of Myh3-positive cells
(P = 0.078), while the delay in growth in the gastrocnemius (GAS)
(B) was statistically significant (interaction: P,0.05). The soleus
(SOL) exhibited no trend for a delay in growth of Myh3-positive
cells (P = 0.42).
(TIF)
Table S1 Pax7-positive cell density in muscle groups ofuntreated and PAI-039 treated WT and Akita mice. TA,gastrocnemius and soleus muscles were analyzed for Pax7-
expressing cells in the regenerating, necrotic and undamaged
regions of CTX-injected skeletal muscles. Data are presented as
mean (6SEM). Untreated group received no treatment, while WT
mice in the ‘‘Vehicle/PAI-039’’ group received vehicle and Akita
mice received PAI-039 treatment. * denotes groups where n= 2.
(DOCX)
Acknowledgments
The Pax7 monoclonal antibody developed by [A. Kawakami] and the
F1.652 monoclonal antibody [developed by H.M. Blau] were obtained
from the Developmental Studies Hybridoma Bank developed under the
auspices of the NICHD and maintained by The University of Iowa,
Department of Biology, Iowa City, IA 52242.
Author Contributions
Conceived and designed the experiments: MPK TJH. Performed the
experiments: MPK DMD DA-S IAR JM TJH. Analyzed the data: MPK
DMD DA-S IAR TJH. Contributed reagents/materials/analysis tools:
MCR TJH. Wrote the paper: MPK MCR TJH.
Figure 6. Macrophage and satellite cell infiltration into necrotic Akita diabetic muscle is restored with PAI-039 treatment. (A)Representative images of the necrotic area of the TA muscles of WT, Akita and Akita+PAI-039. Sections were stained with F4/80 (macrophage marker;red), collagen type 1 (green) and DAPI (nuclear dye; blue). A visible decrease in the number of F4/80 positive cells is seen in Akita muscle sectionswith a detectable increase in F4/80 cells in Akita TA muscles of mice treated with a PAI-1 inhibitor (PAI-039). (B) The number of F4/80 positive cellswithin the necrotic area of injured Akita TA muscles was returned to WT levels when these mice were treated with PAI-039. The number of F4/80positive cells was greater in Akita+PAI-039 TA (panel B; P = 0.06) and gastrocnemius (panel C; P = 0.07) muscles compared to untreated Akita animals.A similar trend was observed for Pax7 positive cells which were also found to be improved in necrotic TA (D) and gastrocnemius (E) with PAI-039treatment (P,0.05).doi:10.1371/journal.pone.0070971.g006
Muscle-Specific Effects in Type 1 Diabetes
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